Cryptic Taxa Revealed through Combined Analysis of Chromosomes and DNA Barcodes: The Polyommatus ripartii Species Complex in Armenia and NW Iran

Simple Summary In the species-diverse butterfly genus Polyommatus, speciation is often driven by rapid changes in chromosome number and structure, resulting in cryptic species. These cryptic species are morphologically similar, but usually can be recognized relatively easily using chromosomal markers. In this work, we show that similar chromosome numbers can independently evolve in different phylogenetic lineages, resulting in species that are difficult to distinguish using routine cytogenetic techniques. We also demonstrate that the combined analysis of chromosomes and mitochondrial DNA barcodes is a simple and effective tool for identifying such cryptic species. Abstract The detection of cryptic species in complexes that have undergone recent speciation is often difficult, since many standard nuclear markers have not yet accumulated differences between closely related taxa, and differences in mitochondrial markers can be leveled out due to mitochondrial introgressions. In these cases, the use of derived chromosomal characters such as non-ancestral chromosomal numbers and/or unusual karyotype features may be a solution to the species delimitation problem. However, non-ancestral but similar karyotypes may arise secondarily as a result of homoplastic evolution, and their interpretation as homologies may lead to incorrect taxonomic conclusions. In our study, we show that the combined use of mitochondrial DNA barcodes and karyotypes helps to solve this problem and identifies cryptic species in situations where each of these markers does not work individually. Using this approach, we show that the fauna of Armenia and adjacent Iran includes the following cryptic taxa of the Polyommatus ripartii species complex (haploid chromosome number, n in parentheses): P. ripartii paralcestis (n = 90), P. ripartii kalashiani, subsp. nov (n close to 90), P. emmeli, sp. nov. (n = 77–79), P. keleybaricus, sp. nov. (n = 86), P. demavendi belovi (n = 73–75), P. demavendi antonius, subsp. nov. (n = 71–73), P. admetus anatoliensis (n = 79) and P. eriwanensis (n = 29–34). Polyommatus admetus yeranyani is synonymized with P. admetus anatoliensis.


Introduction
Despite debates about species concepts and species criteria in biology [1], most scientists agree that speciation is complete when the divergence of evolutionary lineages results in their actual or potential ability to coexist in the same territory without mixing [2][3][4].This ability to coexist is achieved through genetically determined physiological incompatibility, the separation of ecological niches, differences in reproductive behavior, or a combination of these mechanisms [5].As a rule, species, even closely related ones, have fixed morphological differences, which arise either as a by-product of divergent evolution, or as an effect of the direct selection on increasing differences between species [5].However, this is not always the case; species can be cryptic, that is, represent morphologically indistinguishable or highly similar biological entities [6][7][8][9][10][11][12][13][14][15].
Two different processes lead to the formation of cryptic species.Firstly, species are often formed in allopatry, when long-term evolution under conditions of geographic isolation results in postzygotically isolated, but morphologically similar lineages [16].Secondly, speciation is often driven by the formation of prezygotic reproductive isolation, based on differences in ecology or behavior.However, the niche partitioning and recognition of conspecifics on the basis of non-visual mechanisms (for example, differences in acoustic signals and patterns of precopulatory behavior) do not necessarily promote morphological differentiation [17,18].
Genetic markers, such as DNA nucleotide substitutions and karyotype features, are the most reliable and universal characteristics used for the delimitation and identification of cryptic species [10,[19][20][21][22].Each of these two groups of markers has its own specifics, as well as its own advantages and disadvantages.Molecular markers, due to the more uniform dynamics of their evolutionary changes, are better suited to a general assessment of genetic divergence between taxa, which is important for the interpretation of the studied lineages in terms of different taxonomic ranks (genera, tribes, subfamilies, families, etc.) [23].Chromosomal characteristics are often associated with (or even directly cause) postzygotic reproductive isolation.Therefore, they are more convincing as arguments for substantiating species-level taxonomic hypotheses [20,24].It has long been believed that molecular markers (nucleotide substitutions) are more universal characteristics that can be used for substantiating taxonomic and phylogenetic hypotheses, since molecular differences between taxa are always present, and chromosomal differences are present only in limited numbers of cases [25].However, this point of view can be considered outdated.Modern methods of karyotype analysis, based on chromosome-level genome assemblies, show that even homosequential species (i.e., species with identical karyotypes as identified by methods of light microscopy) differ in terms of multiple changes in the structure of their chromosomes.Therefore, de facto chromosomal rearrangements are universal characteristics used for solving problems of systematics and phylogenetics at all taxonomic levels, from the separation of closely related species and the description of subspecies to the analysis of phylogenetic relationships between phyla [26][27][28][29][30].
In real taxonomic practice, the possibilities of using molecular and chromosomal markers are limited by numerous technical, logistical and financial difficulties.If obtaining whole genome data and chromosome-level genome assemblies has become routine for species and populations from Western Europe, North America and East Asia [31,32], this is not so easy for the rest of the world.The delivery to a modern molecular laboratory of the fresh or freshly frozen (−80 • C) samples needed to obtain chromosome-level genome assemblies is practically impossible for numerous rare and undescribed species from Africa, South Asia and Siberia.Therefore, the simplest methods, such as DNA barcoding and routine karyotype analysis, remain the main avenues for obtaining primary genetic information about taxa.The combination of these two tools can have a strong synergistic effect [33,34].
In this study, we used the combined analysis of chromosomes and DNA barcodes to examine the taxonomic structures of blue butterflies of the so-called monomorphic complex, part of the mega-diverse genus Polyommatus Latreille, 1804 [35][36][37][38][39][40][41][42][43][44].The complex received this name because, unlike other species of the genus, which are characterized by strong sexual dimorphism (males have blue wings; females have brown wings), both male and female representatives of the monomorphic complex have a similar brown color [33].Almost all of the numerous species of this complex are cryptic taxa that are indistinguishable by coloration, wing pattern and genital structure, but can be distinguished by karyotypes and molecular markers [33,44,45].
Most species of this complex live in the southern part of the Western Palearctic, from Spain in the west to Iran in the east [45][46][47][48][49][50][51][52][53][54][55].One species, P. ripartii (Freyer, 1830), has a wider distribution, covering the territories of Europe, Asia Minor, Central Asia, Southwest Siberia and West Mongolia [56][57][58][59].Taxonomic revisions have recently been published relating to the species of this complex found in Western Europe [60], the Balkan Peninsula [61], Turkey [62] and Azerbaijan [33].However, the taxa from the more eastern and southern territories remain poorly studied [33].In this paper, based on collections and field studies made over 30 years, we present a taxonomic analysis of the species inhabiting Armenia and adjacent parts of Northwest Iran.

Analysis of Karyotype
Testes were removed from the male abdomens within 1 h of collection and were placed in a 3:1 fixative (3 parts 96% ethanol and 1 part glacial acetic acid).The testes were stored in this fixative for 1-4 weeks at 15-30 • C, and then for 1-10 years at −20 • C. The testes were stained in a saturated solution of orcein (Sigma, Steinheim, Germany) in 45% acetic acid for 7-20 days at 20 • C. We used a two-phase method of chromosome analysis, as described in [61].First, the stained testes were placed in a 40% solution of lactic acid to soften them.The testicular membrane was ruptured using dissecting needles to remove the spermatocysts.The intact spematocysts with unsquashed metaphase plates were analyzed to count the numbers of bivalents in the first metaphase of meiosis or chromosomes in the second metaphase of meiosis.Then, the spermatocysts were squashed using light, gradually increasing pressure on the coverslip.This was achieved by pressing the index finger onto the coverslip and was monitored visually using a 20× and 40× microscope lens before applying immersion oil.This technique led to the fact that chromosomes and bivalents changed their position relative to each other, revealing cases of contact and overlap between chromosomes and bivalents.
The size (area) of bivalents was assessed visually (approximately, "by eye").Although it is technically possible to calculate the area of bivalents in photographs in microns or pixels, such an "exact" calculation does not make sense for three reasons.First, the boundaries of bivalents in a microscope and in photographs are more of a blurred halo than an exact clear line.Second, the size of the bivalents depends to some extent on the saturation of the bivalents with the dye (orcein) during the staining process, and small chromosomes (bivalents) tend to swell more during the staining process than large chromosomes.The process of the saturation of bivalents with orcein is very difficult to control, since it depends on the quality of fixation of the material, which is different in the deep and superficial layers of the testis, and on the ages of the samples, which were different in our study.Third, the bivalent area is a projection onto the plane of a three-dimensional body with a complex configuration.It depends on the orientation of the bivalent in space.Despite these problems, it is clear from the chromosome preparations that in some cases the sizes of the bivalents differ markedly.Therefore, it was decided to evaluate the sizes of bivalents not quantitatively, but qualitatively, using four gradations: (1) the sizes are approximately the same, (2) one bivalent is approximately one and a half times (1.5 times) larger than the other, (3) one bivalent is larger than the other by more than one and a half times and less than two times (indicated as 1.5-1.8times larger), and (4) one bivalent is twice as large.
A Leica DM2500 light microscope (Leica Microsystems, Wetzlar, Germany) equipped with an HC PL APO 100×/1.4Oil CORR CS lens and S1/1.4 oil condenser head was used for bright-field microscopy analysis.The Leica lens HC PL APO 100×/1.40Oil PH3 was used for the phase-contrast microscopy analysis.

Molecular Analysis
Standard mitochondrial DNA barcodes (658 bp COI gene fragments) were obtained for 64 samples (Appendix A) at the Canadian Center for DNA Barcoding (CCDB, Ontario Biodiversity Institute, University of Guelph) and at the Karyosystematics Department of the Zoological Institute of the Russian University, Academy of Sciences.The studied specimens are stored at the Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia.
At the Canadian DNA Barcoding Centre, samples were processed according to the protocols described in [63][64][65].At the Zoological Institute of the Russian Academy of Sciences, samples were processed according to the protocols described in [43,61].For DNA amplification, standard mitochondrial barcode primers LepF1 and LepR1 were used [66,67].All new COI sequences were deposited in GenBank.
The 64 COI sequences obtained in this study (Appendix A) and 63 published COI sequences representing the P. ripartii species complex and the outgroup (P.icarus (Rottemburg, 1775)) were aligned using the BioEdit software [68], resulting in 658 bp alignment (Supplementary File, Table S1).The Bayesian phylogenetic analysis was performed as previously described [61].Briefly, the program MrBayes 3.2 [69] was used with default settings.Two runs of 10,000,000 generations with four chains (one cold and three heated) were performed.The first 25% of each run was discarded as burn-in.The consensus of the obtained trees was visualized using FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/), accessed on 18 October 2023).The minimum COI p-distances (%) between the taxa of the taxa were calculated using the MEGA 11 program [70].

Morphology
Butterfly photographs were taken with a Nikon D810 digital camera (Nikon Corporation, Minato City, Tokyo, Japan) equipped with a Nikon AF-S Micro Nikkor 105 mm lens, using the built-in flash as a lighting source.

Karyotypes
In this study, 48 specimens and eight taxa (species and subspecies) were karyotyped (Table 1, Figure 1).Additionally, 14 specimens of P. eriwanensis (Forster, 1960) karyotyped previously [71] were used for the analysis (Table 1).The chromosome numbers of all the taxa covered in this study, based on new data and previous results, are summarized in Table 2.     3.1.1.Polyommatus ripartii (Figure 1A) At the MI stage, 90 chromosome bivalents were observed in the studied specimen 022K19 from Tajikistan.Of these, two bivalents can be classified as very large bivalent 1 and medium-sized bivalent 2. The largest first bivalent was approximately 1.5-1.8times larger than the medium-sized bivalent 2. The smaller bivalents, from 3 to 90, were found to form a gradient size row in which the largest element 3 was approximately two to three times larger than the smallest element.The largest bivalents 1 and 2 were always observed in the center of metaphase plates.
A similar karyotype was found in samples from Armenia (Gyumarants and Ja Joor pass), but the number of chromosomes was determined to be approximately equal to 90, due to the fact that it was not possible to find metaphase plates of ideal quality without overlapping and touching chromosomes.A variable number (from n = 77 to n = 79) of distinct chromosome elements were found at the MI and MII cells of the studied 14 specimens.In structure (the number of large bivalents and their relative sizes), the karyotype was similar to that of P. ripartii.There are two large elements in the set.At the MI stage, the first and largest bivalent was approximately 1.5-1.8times larger than the medium-sized bivalent 2. The remaining smaller bivalents were found to form a gradient size row in which the largest element 3 was approximately two to three times larger than the smallest element.The largest bivalents 1 and 2 were always observed in the center of metaphase plates.
3.1.3.Polyommatus keleybaricus, sp.nov.(Figure 1D,E) The haploid chromosome number was identified to be n = 86 based on analyses of five samples (Table 1).In terms of the structure of the MI metaphase plates (the number of large bivalents and their relative sizes), the karyotypewas similar to that of P. ripartii.There are two large elements in the set.At the MI stage, the largest first bivalent was approximately 1.5-1.8times larger than the medium-sized bivalent 2. The remaining smaller bivalents were found to form a gradient size row in which the largest element 3 was approximately two to three times larger than the smallest element.The largest bivalents 1 and 2 were always observed in the center of metaphase plates.
3.1.4.Polyommatus admetus yeranyani (Dantchenko et Lukhtanov, 2004) (Figure 1F) The haploid chromosome number was identified to be n = 79 based on analyses of five samples (Table 1).In the sample KL-34-96 from Armenia (Aragats), the haploid chromosome number was identified to be close to n = 80; however, this count was made with an approximation due to the fact that it was not possible to find metaphase plates of ideal quality without overlapping and touching chromosomes.Bivalents at the MI stage and chromosomes at the MII stage are highly differentiated in size, and one of the largest element, located in the center, is always easily recognized.All other elements, from medium to very small, form a gradient size row, within which the individual identification of chromosomes is impossible.Thus, this karyotype differs from that of P. emmeli, which also has a haploid chromosome number of n = 79.
3.1.5.Polyommatus demavendi belovi (Dantchenko et Lukhtanov, 2004) (Figure 1G,H) A variable number (from n = 73 to n = 75) of distinct chromosome elements were found at the MI and MII cells of the studied 12 specimens.In terms of structure (the number of large bivalents and their relative sizes), the karyotype is distinct if compared with karyotypes of P. ripartii, P. emmeli, P. keleybaricus and P. admetus.There are two large and two medium-sized elements in the set.At the MI stage, the two largest bivalents were approximately 1.5 times larger than the two medium-sized bivalents.The remaining smaller bivalents were found to form a gradient size row.The two largest bivalents 1 and 2 and the two medium-sized bivalents 3 and 4 were always observed in the center of metaphase plates.
Insects 2024, 15, 545 8 of 24 3.1.6.Polyommatus demavendi antonius, subsp.nov.(Figure 1I) A variable number (from n = 71 to n = ca73) of distinct chromosome elements were found at the MI and MII cells of the six studied specimens.In terms of structure (the number of large bivalents and their relative sizes), the karyotypeis similar to that of Polyommatus demavendi belovi.There are two large and two medium-sized elements in the set.At the MI stage, the two largest bivalents were approximately 1.5 times larger than the two mediumsized bivalents.The remaining smaller bivalents were found to form a gradient size row.The two largest bivalents 1 and 2 and the two medium-sized bivalents 3 and 4 were always observed in the centers of metaphase plates.

Phylogenetic Analysis and Clustering of the Studied Samples by COI Haplotypes and Karyotypes
The Bayesian analysis (Figure 2) identified P. eriwanensis as a highly differentiated clade, sister to the lineage containing the remaining taxa.Within the latter lineage, specimens of P. admetus (Esper, 1783) from eastern Turkey (P.admetus anatoliensis (Forster, 1960)), Iran, Armenia (P.admetus yeranyani (Dantchenko et Lukhtanov, 2004)), and Azerbaijan (P.admetus malievi (Dantchenko et Lukhtanov, 2004)) formed a basal polytomy.Specimens of P. admetus admetus from southeastern Europe and western Turkey were identified as a discrete lineage differentiated from P. admetus anatoliensis (including P. admetus yeranyani) that appeared on the tree as a paraphyletic assemblage.In should be noted that the posterior supports for this paraphyletic topology were low and not inconsistent with the monophyly of P. admetus anatoliensis if markers with stronger phylogenetic signal are used.
The studied samples from Armenia and the adjacent territory of Iran (highlighted by colors on the phylogeny in Figure 2) were found to form seven differentiated groups of individuals.Four of these groups (1, 2, 4 and 7) are highly supported monophyletic lineages.The groups 3, 5 and 6 appeared on the DNA barcode tree as paraphyletic assemblages, although their monophyly cannot be excluded and could theoretically be justified in the future using markers with a stronger phylogenetic signal.
Group 2 (P.ripartii kalashiani) is monophyletic with respect to the COI gene.This group is characterized by a haploid number of chromosomes n = ca90; in the set there is one large and one medium bivalent.This group has a karyotype indistinguishable from that of P. ripartii paralcestis and P. ripartii ripartii (Table 2).
Group 3 (P.emmeli) has DNA barcodes indistinguishable from those of P. riparti paralcestis, but this group has a unique karyotype distinctly different from the karyotype of P. ripartii.It is characterized by haploid chromosome numbers n = 77-79; the set contains one large and one medium bivalent, distinguishing it from P. riparti paralcestis (n = 90) (Table 2).
Group 4 (P.keleybaricus) is monophyletic with respect to the COI gene.It has a unique karyotype (n = 86) (Figure 1D, E).
Group 5 (P.demavendi belovi) has DNA barcodes indistinguishable from those of P. demavendi demavendi from Iran and P. demavendi amasyensis from Turkey, but has a higher number of chromosomes than P. demavendi demavendi and P. demavendi amasyensis (Table 2).
Group 7 (P.eriwanensis) is monophyletic with respect to the COI gene.It is characterized by haploid chromosome numbers n = 29-34 (Tables 1 and 2).
It should be noted that representatives of the groups 3 (P.emmeli), 5 (P.demavendi belovi), 6 (P.admetus yeranyani) and 7 (P.eriwanensis) were found in sympatry in Gnishik (Table 1).The studied samples from Armenia and the adjacent territory of Iran (highlighted by colors on the phylogeny in Figure 2) were found to form seven differentiated groups of individuals.Four of these groups (1, 2, 4 and 7) are highly supported monophyletic lineages.The groups 3, 5 and 6 appeared on the DNA barcode tree as paraphyletic assemblages, although their monophyly cannot be excluded and could theoretically be justified in the future using markers with a stronger phylogenetic signal.

Color and Wing Pattern
All studied butterflies are indistinguishable by the upper side of the wings, which have a dark brown color in males with a dense network of androconial scales (Figures 3 and 4).The underside is characterized by the presence of a white stripe on the hind wings and a system of basal, discal and postdiscal spots, as well as a marginal pattern, the intensity of which varies to the greatest extent from almost complete reduction (Figure 3A) to a high degree of intensity Figures 3B-E and 4G).A more detailed analysis of this figure is given below when describing the species and subspecies.
It would be tempting to treat all these clusters as taxa, but discrete genetic differences between individuals are not yet evidence that they belong to different species [23].Groups of individuals, discrete by genetic markers, may represent either different taxa, or variants of intraspecific genetic polymorphism.We previously proposed general principles for the interpretation of such clusters, based on the joint use of karyotype characteristics and DNA barcodes [33].The chromosomal characters and the mitochondrial DNA barcodes represent two different, not physically linked parts of the genome, namely, nuclear (karyotypes) and mitochondrial (DNA barcodes) markers.In the case of intra-population variability, the linkage equilibrium of nuclear and mitochondrial markers is expected [34].This indicates the situation in which the markers combine at random and form all possible combinations of mitochondrial and chromosomal characteristics.Additionally, in the case of intra-population variability, we should expect numerous chromosomal heterozygotes that can be recognized via tri-and multivalents observed at the first meiotic metaphase [20,33,61].In the case of two cryptic species, hybridization between the clusters is absent or very limited.Therefore, we should expect a situation in which nuclear and mitochondrial genes mimic linkage disequilibrium, forming stable species-specific combinations [33].In addition, in the case of cryptic species, the absence of interspecific hybridization leads to the absence of heterozygotes for chromosomal rearrangements [20,33,61].
In our study, the latter situation was found in Gnishik (south slopes of Vayots Dzor range, Armenia), where we discovered four clusters (eriwanensis, admetus, demavendi belovi and emmeli) in complete sympatry (Figure 5).Of these four clusters, three (eriwanensis, admetus, and demavendi belovi) were described previously as taxa [33,71], and the fourth cluster is described in this work below as a new species, P. emmeli, sp.nov.Within these four sympatric clusters, each entity is characterized by a stable combination of a specific mitochondrial haplogroup and a unique karyotype.During our study, we also did not find chromosomal heterozygotes that are expected to exist in the case of interspecific hybridization.All this indicates that all four clusters, including the previously unknown P. emmeli, belong to different biological species.
When the clusters are allopatric, the direct application of the species criteria based on the limited interspecific hybridization is not possible.Therefore, two groups of individuals living in allopatry should be considered different species if they are differentiated so strongly that reproductive isolation between them can be assumed, or if their merging would result in non-monophyletic assemblage [33,34].The presence of fixed chromosomal rearrangements between two or more groups of individuals, as a rule, indicates their non-conspecificity [20,44,45,[59][60][61], since usually even relatively small differences in karyotypes lead to disorders of meiosis and hybrid sterility [72].Nevertheless, in some cases, differences in karyotypes do not lead to postzygotic isolation [73].In particular, in Lepidoptera, there are cases of fertility in hybrids between chromosomally divergent races [73], as well as high intraspecific variability in the number of chromosomes [44,45].The latter phenomenon is incompatible with strong selection against chromosomal heterozygotes.However, it should be borne in mind that even in this case, chromosomal differences affect reproductive isolation, suppressing recombination in rearranged chromosome regions in hybrids [74][75][76][77][78].When the clusters are allopatric, the direct application of the species criteria based on the limited interspecific hybridization is not possible.Therefore, two groups of individuals living in allopatry should be considered different species if they are differentiated so strongly that reproductive isolation between them can be assumed, or if their merging would result in non-monophyletic assemblage [33,34].The presence of fixed chromosomal rearrangements between two or more groups of individuals, as a rule, indicates their nonconspecificity [20,44,45,[59][60][61], since usually even relatively small differences in karyotypes lead to disorders of meiosis and hybrid sterility [72].Nevertheless, in some cases, differences in karyotypes do not lead to postzygotic isolation [73].In particular, in Lepidoptera, there are cases of fertility in hybrids between chromosomally divergent races [73], as well as high intraspecific variability in the number of chromosomes [44,45].The showing the distribution of P. ripartii paralcestis (black), P. ripartii kalashiani (grey), P. emmeli (red), P. keleybaricus (violet), P. demavendi belovi (blue), P. demavendi antonius (brown), P. eriwanensis (yellow), and P. admetus anatoliensis (green).In (B), 1 is Armenia, 2 is Azerbaijan (Nakhchivan), and 3 is Azerbaijan.The size of the circle corresponds to the number of species found in the locality (one, two or four species).
The criterion of chromosomal differences can be used for the taxonomic interpretation of the allopatric taxa P. riparii paralcestis (n = 90), P. emmeli (n = 77-79) and P. keleybaricus (n = 86), which differ in chromosomal numbers.All these three taxa have similar mitochondrial DNA barcodes, which indicates either the origin of these taxa from a common ancestor, or relatively recent mitochondrial introgression.A comparative phylogenetic analysis of the accumulation of chromosomal rearrangements leading to changes in chromosome numbers in the genus Polyommatus shows that chromosome numbers evolve slowly, and there is a significant correlation between chromosome number changes and phylogeny branch lengths [79].Therefore, given that mitochondrial introgression is common in different organisms, including Lepidoptera [80][81][82][83][84][85][86][87], the DNA barcode identity between deeply differentiated chromosomal lineages appears to be a consequence of mitochondrial introgression rather than the ancestrality of this character.This hypothesis should be tested in future via analyses of nuclear markers that are expected to be differentiated.Therefore, we believe that fixed differences between the karyotypes of P. riparii paralcestis (n = 90), P. emmeli (n = 77-79) and P. keleybaricus (n = 86) allow us to interpret them as different species.
The allopatric taxa P. demavendi demavendi, P. demavendi amasiensis, P. demavendi lorestanus and P. demavendi belovi [33,88], and the population from the Lake Sevan region, which will be described below as P. demavendi antonius, have similar chromosome numbers and an identical karyotype structure (a combination of two large and two medium chromosomes), which can be interpreted as a chromosomal synapomorphy.On this basis, they can be combined into one taxon of species rank (P.demavendi), despite the fact that the monophyly of this group is unresolved on the tree based on its short DNA barcodes.
Within this species, two subspecies (P.d. lorestanus and P. d. antonius) are well differentiated by mitochondrial DNA and represent distinct phylogenetic sublineages.As for P. d. belovi and P. d. demavendi, they show differences in chromosome numbers, which are close and variable within each group (n = 67-72 in P. d. demavendi and n = 73-75 in P. d. belovi), but do not overlap [33,88].Thus, the combined molecular and chromosomal data provide evidence of the subspecies status of P. demavendi demavendi, P. demavendi lorestanus, P. demavendi belovi and P. demavendi antonius.
The allopatric taxa P. ripartii paralcestis and P. ripartii kalashiani are also differentiated by DNA barcodes, and have clear differences in the wing color.These taxa can be interpreted as subspecies, if by subspecies we mean differentiated phylogeographic sublineages [89].
In our study, we found a lack of differentiation by DNA barcodes between P. admetus anatoliensis (central Turkey) and P. admetus yeranyani (Armenia).The karyotypes of these two taxa are also identical.Thus, these nominal taxa do not represent phylogeographic sublineages, which is essential for justifying subspecific status [89].As for morphology, butterflies with a pronounced marginal pattern on the underside of the hindwings predominate in central Turkey.In Armenia and adjacent regions of Iran, butterflies with this phenotype are also found (Figure 4G); however, individuals with a reduced marginal pattern predominate (Figure 4F).Thus, no fixed differences were found between populations from central Turkey and Armenia.On this basis, the taxon P. admetus yeranyani, syn nov. is synonymized here with P. admetus anatoliensis.
The subspecies P. admetus malievi, found in the easternmost part of the species' range, also does not differ from P. admetus anatoliensis in karyotype [33].Its monophyly for the COI gene has not been proven in our study due to the low support for the cluster formed by individuals of this subspecies.However, the difference in phenotypes between P. admetus malievi and P. admetus anatoliensis is substantial, and we do not have material showing the clinal morphological transition from P. admetus malievi to P. admetus anatoliensis.On this basis, we propose to maintain the subspecies status of P. admetus malievi.populations from central Turkey and Armenia.On this basis, the taxon P. admetus yeranyani, syn nov. is synonymized here with P. admetus anatoliensis.The subspecies P. admetus malievi, found in the easternmost part of the species' range, also does not differ from P. admetus anatoliensis in karyotype [33].Its monophyly for the COI gene has not been proven in our study due to the low support for the cluster formed by individuals of this subspecies.However, the difference in phenotypes between P. admetus malievi and P. admetus anatoliensis is substantial, and we do not have material showing the clinal morphological transition from P. admetus malievi to P. admetus anatoliensis.On this basis, we propose to maintain the subspecies status of P. admetus malievi.Males (Figure 3F,G).Forewing length in holotype 15.1 mm.Forewing length in paratypes 14.9-16.8mm.

Polyommatus emmeli
Upperside.Ground color is dark brown with slightly darker veins.Discoidal, submarginal and antemarginal marking is absent on both fore-and hindwings.Forewings have good developed sex brands and scaletufts.Fringe is brown, as is ground color.
Underside.Ground color is light greyish brown.Greenish blue basal suffusion on the hindwings is slightly visible.Discoidal black spots are present on forewings.Postdiscal black marking is small or even reduced on hindwings and or medium-sized on fore wings, circled with white on both fore-and hindwings.Submarginal markings are well pronounced on the hindwing, antemarginal markings are presented by fuzzy lunulae.On the forewings, sub-and antemarginal markings are strongly reduced, practically absent.White streak on hindwings underside clearly visible, enlarged distally.Fringe light brown, slightly darker than underside ground color.
Genitalia.In males, the genitalia have the same structure as in other species of the Polyommatus subgenus Agrodiaetus Hübner, 1822 [90].We did not find any species-specific features in their structure.Briefly described, it can be noted that the uncus consists of two sclerotized parts.Gnathos has the appearance of sclerotized hooks.Yuxta has two long narrow branches.The relatively short aedeagus is straight, not curved.The lengths of the valvae are more than four times their width.There is a convex membranous fold on the ventral surface of the valva.The sacculus is well developed and extends along the entire ventral margin of the valvae.
Females.Although we have collected a significant number of females that are highly likely to belong to this species, we choose not to describe them here for the following reasons.Reliable identification of the species using DNA barcodes is impossible, since it shares barcodes with P. ripartii.Polyommatus ripartii is not found in Gnishik and Sevan, where the new species is described; however, it is found in adjacent territories, and we cannot completely exclude its sympatry with P. emmeli.The reliable identification of P. emmeli is possible by karyotype, but methods for analyzing female karyotypes for the genus Polyommatus have not been developed.Therefore, the description of females will only be possible after they are collected in copula with males, if the karyotype of these males is studied.
Diagnosis.Polyommatus emmeli can be reliably differentiated from other species by its chromosome number of n = 77-79, in combination with the presence of two marker chromosomes, the largest in the set and the middle one, which is noticeably smaller than the first, but substantially larger than the third and subsequent ones in the set.It differs from the sympatric P. admetus, which also has n = 79, by its karyotype structure (P.admetus Onobrychis radiata Bieberstein, 1810 (Section Heliobrychis), which is most probably a larval food plant for P. demavendi antonius.The butterflies are on their wings from the end of June.Ecologically, P. demavendi antonius differs from P. demavendi belovi, which inhabits dry biotopes often on gypsum soils in lowland desert-like areas.The larval food plant of P. demavendi belovi is Onobrychis atropatana Boiss.
Distribution.Northern Armenia (Lake Sevan region) (Figure 5A).Etymology.Named in honor of Anton Dantchenko, who helped in collecting and fixing the butterflies on our trip to Lake Sevan.color with grayish tint on the underside of the wings.From P. demavendi belovi, which occurs in close proximity in the more southern parts of Armenia, the new subspecies differs in the large number of chromosomes, indicating the presence of one or even two fixed chromosomal fusions/fissions separating these subspecies.Bionomy.Polyommatus demavendi antonius inhabits xerophilic calcerous slopes with tragacanth vegetation in the juniper woodland belt from 2100 to 2400 m above sea level.The last-instar larvae were collected from the end of May until the middle of June on Onobrychis radiata Bieberstein, 1810 (Section Heliobrychis), which is most probably a larval food plant for P. demavendi antonius.The butterflies are on their wings from the end of June.Ecologically, P. demavendi antonius differs from P. demavendi belovi, which inhabits dry biotopes often on gypsum soils in lowland desert-like areas.The larval food plant of P. demavendi belovi is Onobrychis atropatana Boiss.Distribution.Northern Armenia (Lake Sevan region) (Figure 5A).Etymology.Named in honor of Anton Dantchenko, who helped in collecting and fixing the butterflies on our trip to Lake Sevan.Upperside.The ground color is dark brown with slightly darker veins.Discoidal, submarginal and antemarginal markings are absent on both fore-and hindwings.Forewings have good developed sex brands and scaletufts.The fringe is brown, as is the ground color.In general, the uppersides of the wings are no different from those of the other studied taxa.Underside.Ground color is greyish brown.Greenish blue basal suffusion on the hindwings is clearly visible.Basal black spots are present only on hindwings.Discoidal black spots are present on forewings.Postdiscal black spots are relatively large on both Males (Figure 3B,C).The forewing length in the holotype is 16.9 mm.The forewing length in paratypes is 16.5-17.0mm.

Polyommatus ripartii kalashiani
Upperside.The ground color is dark brown with slightly darker veins.Discoidal, submarginal and antemarginal markings are absent on both fore-and hindwings.Forewings have good developed sex brands and scaletufts.The fringe is brown, as is the ground color.In general, the uppersides of the wings are no different from those of the other studied taxa.
Underside.Ground color is greyish brown.Greenish blue basal suffusion on the hindwings is clearly visible.Basal black spots are present only on hindwings.Discoidal black spots are present on forewings.Postdiscal black spots are relatively large on both fore-and hindwings, circled with white on both fore-and hindwings.The submarginal marking is very well pronounced on the hindwing, and the antemarginal marking is also present.On the forewings, sub-and antemarginal markings are less pronounced than on the forewing, but are nevertheless clearly present.The white streak on the hindwings' underside is bright, enlarged distally.Fringe is light brown, slightly darker than underside ground color.
Genitalia: the male genitalia have a structure typical for other species of the subgenus Agrodiaetus [90].
Karyotype.See Results section above (Table 1).Females.Unknown.No females with the keleybaricus mitochondrial haplogroup were found within the studied samples.
Diagnosis.Polyommatus ripartii kalashiani differs from other subspecies of P. ripartii in the strong lightening of the marginal region on the undersides of the fore-and hindwings, against which a contrasting marginal pattern is developed.P. keleybaricus has a similar type of pattern on the underside of the wings; however, in P. keleybaricus, the wings have a lighter gray-brownish color on the underside.It differs from species P. ripartii paralcestis, P. keleybaricus and P. emmeli by the fixed nucleotide substitution A⇔G in the position 420 of the studied COI region.
Bionomy.Polyommatus ripartii kalashiani inhabits dry meadows and clearings on the upper borders of mountain oak forests, from 1400 to 1900 m above sea level.The butterflies are on their wings from mid-July to August.The larvae's food plant is, most probably, an undetermined Onobrychis species (section Onobrychis) [92], which is common in the biotope.
Etymology.Named in honor of Mark Kalashian, our friend and colleague, a prominent Armenian zoologist.

Figure 2 .
Figure 2. The Bayesian tree based on analysis of the mitochondrial COI barcodes.Numbers at nodes indicate Bayesian posterior probabilities (BPP) (higher than 0.5).The number in brackets (after species and subspecies names) refers to the number of analyzed specimens.1-7 are differentiated groups of individuals found in Armenia and the adjacent territory of Iran (highlighted by different colors).The groups 3, 5 and 6 are paraphyletic.Samples from other territories are not highlighted.Scale bar = 0.02 substitutions per position.Groups of individuals with support higher than 0.5 are collapsed to make the Figure 2 more compact.The finer structure of the collapsed groups is shown in Figure S1 in Supplementary Materials.

Figure 2 .
Figure 2. The Bayesian tree based on analysis of the mitochondrial COI barcodes.Numbers at nodes indicate Bayesian posterior probabilities (BPP) (higher than 0.5).The number in brackets (after species and subspecies names) refers to the number of analyzed specimens.1-7 are differentiated groups of individuals found in Armenia and the adjacent territory of Iran (highlighted by different colors).The groups 3, 5 and 6 are paraphyletic.Samples from other territories are not highlighted.Scale bar = 0.02 substitutions per position.Groups of individuals with support higher than 0.5 are collapsed to make the Figure 2 more compact.The finer structure of the collapsed groups is shown in Figure S1 in Supplementary Materials.

Table 1 .
List of the karyotyped samples with their chromosome numbers.

Table 2 .
Chromosome numbers of all the taxa covered in this study, based on new data and previous results.
from Spain to Mongolia and Central Asia [45,59-61], this study Insects 2024, 15, x FOR PEER REVIEW 6 of 26

Table 2 .
Chromosome numbers of all the taxa covered in this study, based on new data and previous results.